scholarly journals Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae

2015 ◽  
Vol 53 (9) ◽  
pp. 2810-2815 ◽  
Author(s):  
Anne Liljander ◽  
Mingyan Yu ◽  
Elizabeth O'Brien ◽  
Martin Heller ◽  
Julia F. Nepper ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Recombinase Polymerase Amplification ◽  
Target Sequence ◽  
Content Type ◽  
Mycoplasma Capricolum ◽  
Contagious Caprine Pleuropneumonia ◽  
Geographical Regions ◽  
Amplification Assay ◽  
Simulated Field ◽  
High Degree

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused byMycoplasma capricolumsubsp.capripneumoniaethat affects goats in Africa and Asia. Current available methods for the diagnosis ofMycoplasmainfection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection ofM. capricolumsubsp.capripneumoniae. We developed the assay using a specific target sequence inM. capricolumsubsp.capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 103and 5 × 104cells/ml were obtained using genomic DNA and bacterial culture fromM. capricolumsubsp.capripneumoniaestrain ILRI181, while no amplification was obtained from 71 relatedMycoplasmaisolates or from theAcholeplasmaor thePasteurellaisolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

scholarly journals Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum

2020 ◽  
Vol 58 (5) ◽  
Author(s):  
Le Jiang ◽  
Philip Ching ◽  
Chien-Chung Chao ◽  
J. Stephen Dumler ◽  
Wei-Mei Ching
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Limit Of Detection ◽  
Dna Amplification ◽  
Recombinase Polymerase Amplification ◽  
Detection Methods ◽  
Content Type ◽  
Amplification Assay ◽  
Nonspecific Amplification

ABSTRACT Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Development of a recombinase polymerase amplification assay forVibrio parahaemolyticusdetection with an internal amplification control

2018 ◽  
Vol 64 (4) ◽  
pp. 223-230 ◽  
Author(s):  
Huan-Lan Yang ◽  
Shuang Wei ◽  
Ravi Gooneratne ◽  
Anthony N. Mutukumira ◽  
Xue-Jun Ma ◽  
...  
Keyword(s):  
Bacterial Species ◽  
False Negative ◽  
Recombinase Polymerase Amplification ◽  
Internal Amplification Control ◽  
Target Sequence ◽  
Negative Results ◽  
False Negative Results ◽  
Amplification Assay ◽  
Pcr Method ◽  
Recombinase Polymerase Amplification Assay

A novel RPA–IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA–IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

scholarly journals Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

2011 ◽  
Vol 77 (13) ◽  
pp. 4329-4335 ◽  
Author(s):  
Bilgin Taskin ◽  
Ayse Gul Gozen ◽  
Metin Duran
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Membrane Integrity ◽  
Dna Amplification ◽  
Live Cells ◽  
Dead Cell ◽  
Target Sequence ◽  
Propidium Monoazide ◽  
Indicator Organisms ◽  
Content Type

ABSTRACTQuantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed withEscherichia coliATCC 25922 as the model organism and theuidAgene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1.

scholarly journals Evaluation of the illumigene Mycoplasma Direct DNA Amplification Assay

2018 ◽  
Vol 56 (7) ◽  
Author(s):  
Neena Kanwar ◽  
Morgan A. Pence ◽  
Donna Mayne ◽  
Jeffrey Michael ◽  
Rangaraj Selvarangan
Keyword(s):  
Direct Detection ◽  
False Negative ◽  
Reference Method ◽  
Dna Amplification ◽  
The United States ◽  
Community Acquired Pneumonia ◽  
Content Type ◽  
Amplification Assay ◽  
Respiratory Specimens

ABSTRACT Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The illumigene Mycoplasma Direct (iMD) DNA amplification assay is a qualitative in vitro test utilizing loop-mediated isothermal amplification (LAMP) technology for the direct detection of M. pneumoniae DNA in respiratory specimens. The iMD assay does not require the preextraction of nucleic acids from specimens, which is a prerequisite step for the previously approved illumigene Mycoplasma (iM) assay. The aim of this prospective multicenter study was to evaluate the performance characteristics of the newly developed iMD assay, compared with the iM assay. Subjects with symptoms of upper respiratory illnesses suggesting M. pneumoniae infection were enrolled at three sites in the United States. Respiratory specimens were obtained using dual throat swabs. One swab was tested with the iMD assay at each enrollment site. Reference testing with the iM assay was performed by the manufacturer. Among 456 specimens tested, the iM reference method detected M. pneumoniae in 25 specimens (5.5%), while the iMD assay identified 34 specimens (7.5%) as M. pneumoniae positive. There were 10 false-positive results and 1 false-negative result with the iMD assay. The overall positive and negative agreement rates were 96.0% (95% confidence interval [CI], 80.5 to 99.3%) and 97.7% (95% CI, 95.8 to 98.7%), respectively. The overall agreement rate was determined to be 97.6% (95% CI, 95.7 to 98.6%). We conclude that the iMD test results were comparable to the iM assay results. The removal of the DNA extraction step for the iMD assay simplifies testing, saves time, and reduces the costs of detecting M. pneumoniae from throat swabs, compared to the iM assay.

scholarly journals Complete Genome Sequence of Mycoplasma capricolum subsp. capripneumoniae Strain zly1309F, Isolated from Endangered Tibetan Antelope

2017 ◽  
Vol 5 (29) ◽  
Author(s):  
Huafang Hao ◽  
Shengli Chen ◽  
Yuanguo Li ◽  
Heting Sun ◽  
Ping Zhao ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Content Type ◽  
Mycoplasma Capricolum ◽  
Contagious Caprine Pleuropneumonia ◽  
Tibetan Antelope

ABSTRACT Mycoplasma capricolum subsp. capripneumoniae is an important pathogen of goats that causes contagious caprine pleuropneumonia. Here, we report the complete genome sequence of M. capricolum subsp. capripneumoniae strain zly1309F, isolated from a Tibetan antelope (Pantholops hodgsonii) in China.

scholarly journals Greater fit and a greater gap

2019 ◽  
Vol 26 (4) ◽  
pp. 561-594
Author(s):  
Steven A. Brieger ◽  
Dirk De Clercq ◽  
Jolanda Hessels ◽  
Christian Pfeifer
Keyword(s):  
Life Satisfaction ◽  
Institutional Environment ◽  
Entrepreneurial Activity ◽  
Worker Rights ◽  
Content Type ◽  
Person Environment Fit ◽  
High Power Distance ◽  
High Degree ◽  
The Impact ◽  
Low Uncertainty

Purpose The purpose of this paper is to understand how national institutional environments contribute to differences in life satisfaction between entrepreneurs and employees. Design/methodology/approach Leveraging person–environment fit and institutional theories and using a sample of more than 70,000 entrepreneurs and employees from 43 countries, the study investigates how the impact of entrepreneurial activity on life satisfaction differs in various environmental contexts. An entrepreneur’s life satisfaction arguably should increase when a high degree of compatibility or fit exists between his or her choice to be an entrepreneur and the informal and formal institutional environment. Findings The study finds that differences in life satisfaction between entrepreneurs and employees are larger in countries with high power distance, low uncertainty avoidance, extant entrepreneurship policies, low commercial profit taxes and low worker rights. Originality/value This study sheds new light on how entrepreneurial activity affects life satisfaction, contingent on the informal and formal institutions in a country that support entrepreneurship by its residents.

scholarly journals Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Georgios Chondrogiannis ◽  
Shirin Khaliliazar ◽  
Anna Toldrà ◽  
Pedro Réu ◽  
Mahiar M. Hamedi
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Point Of Care ◽  
Dna Amplification ◽  
Recombinase Polymerase Amplification ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Enzymatic Function ◽  
Modern Biotechnology ◽  
Nucleic Acid Tests

AbstractEnzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.

scholarly journals Nationwide Temporal and Geographical Distribution of Tick Populations and Phylogenetic Analysis of Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in Korea, 2020

2021 ◽  
Vol 9 (8) ◽  
pp. 1630
Author(s):  
Min-Goo Seo ◽  
Byung-Eon Noh ◽  
Hak Seon Lee ◽  
Tae-Kyu Kim ◽  
Bong-Goo Song ◽  
...  
Keyword(s):  
Tick Species ◽  
Temporal Distribution ◽  
Epidemiological Studies ◽  
Haemaphysalis Longicornis ◽  
Geographical Regions ◽  
Control And Prevention ◽  
Amblyomma Testudinarium ◽  
High Degree ◽  
Severe Fever ◽  
Minimum Infection Rate

Since 2010, the Korea Disease Control and Prevention Agency has established centers at 16 locations to monitor disease vectors and pathogens. Here, we examined tick populations to understand the geographical and temporal distribution of severe fever with thrombocytopenia syndrome virus (SFTSV) vectors in 2020. From April to November, 63,376 ticks were collected from traps to monitor tick populations, with a trap index of 41.3. Tick incidence varied from April to October, with population peaks observed for nymphs in May, adults in July, and larvae in September. The predominant tick species were Haemaphysalis longicornis, Haemaphysalis spp., H. flava, Ixodes spp., Amblyomma testudinarium, and Ixodes nipponensis. Approximately 50% of the collected ticks were pooled into 2973 groups to detect the rate of SFTSV infection in ticks. The minimum infection rate (MIR) of SFTSV was 0.2%, and Andong had the highest MIR for SFTSV (4.0%). The B3 genotype was the most prevalent (52.2%) followed by B2 (28.6%), B5 (15.9%), B4 (1.6%), and B6 (1.6%). We identified widely distributed tick species and a high degree of diversity in SFTSV strains in ticks from different geographical regions. The results may provide a basis for future epidemiological studies and risk assessments for tick-borne diseases.

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